title: A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.
abstract: Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.
authors.affiliation: Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, 37200 Tours, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Besseau
authors.forename: Franziska
authors.initials: F
authors.lastname: Kellner
authors.forename: Arnaud
authors.initials: A
authors.lastname: Lanoue
authors.forename: Antje M K
authors.initials: AM
authors.lastname: Thamm
authors.forename: Vonny
authors.initials: V
authors.lastname: Salim
authors.forename: Bernd
authors.initials: B
authors.lastname: Schneider
authors.forename: Fernando
authors.initials: F
authors.lastname: Geu-Flores
authors.forename: René
authors.initials: R
authors.lastname: Höfer
authors.forename: Grégory
authors.initials: G
authors.lastname: Guirimand
authors.forename: Anthony
authors.initials: A
authors.lastname: Guihur
authors.forename: Audrey
authors.initials: A
authors.lastname: Oudin
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Glevarec
authors.forename: Emilien
authors.initials: E
authors.lastname: Foureau
authors.forename: Nicolas
authors.initials: N
authors.lastname: Papon
authors.forename: Marc
authors.initials: M
authors.lastname: Clastre
authors.forename: Nathalie
authors.initials: N
authors.lastname: Giglioli-Guivarc'h
authors.forename: Benoit
authors.initials: B
authors.lastname: St-Pierre
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
authors.forename: Vincent
authors.initials: V
authors.lastname: Burlat
authors.forename: Vincenzo
authors.initials: V
authors.lastname: De Luca
authors.forename: Sarah E
authors.initials: SE
authors.lastname: O'Connor
authors.forename: Vincent
authors.initials: V
authors.lastname: Courdavault
mesh_terms: D055162:Biocatalysis; D053898:Biosynthetic Pathways / Q000235:genetics; D030008:Catharanthus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant / Q000235:genetics; D006900:Hydroxylation; D026121:Indole Alkaloids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D055442:Metabolome / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D019441:Plant Epidermis / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D026121:Indole Alkaloids; D010940:Plant Proteins; D011804:Quinolines; D012333:RNA, Messenger; C009373:tabersonine; C009667:vindoline; D014747:Vinblastine; D003577:Cytochrome P-450 Enzyme System; C121596:CYP71D12 protein, Catharanthus roseus